Method of determining the suppressor cell component of human immune status and a means for the realization thereof

ABSTRACT

The method for determining a suppressor component of human immune status comprises collecting peripheral blood, obtaining a suspension of mononuclear cells (MNCs), dividing said suspension into two equal portions, cultivating MNCs in the first portion without a suppressor activator, cultivating MNCs in the second portion with said suppressor activator (TBG), washing MNCs out of culture medium, blocking the proliferation, adding newly isolated MNCs from a normal donor, which MNCs have been stimulated with phytohemagglutinin, into each of MNC portions in equal proportions to obtain test cultures, cultivating said test cultures, further evaluating the proliferation in said test cultures and determining the suppression value on the basis of the ratios of proliferation levels in test cultures.

This application is a national stage entry of PCT/Ru95/00046 filed Mar.16, 1995.

FIELD OF TECHNOLOGY

The present invention relates to medicine, more specifically to themethod of diagnostic evaluation of T-suppressors activity, namely, tothe method for determining a suppressor component of human immune statusand to the means for the realization thereof.

PRIOR ART

Known is β-I-glycoprotein of placental origin which is a oftrophoblastic β-I-glycoprotein (TBG) used as a growth and proliferationstimulator for hematopoietic blood cells (U.S. Pat. No. 5,169,835, cl.A61K 35/50, 1989).

Nevertheless the known compound has not been used for determining asuppressor component of human immune status.

Known is the use of TBG for diagnosing and prognosticating the course ofpregnancy (see L. G. Sotnikova et al., The Role of β-I-glycopzoteinTrophoblast in Diagnosing and Prognosticating Pregnancy, MetodicheskijeRecomendatsii, Moscow, 1984). Nevertheless said work does not disclosethe possibility of using TBG for diagnosing a suppressor component ofhuman immune status.

Known is method for determining a suppressor component of human immunestatus which comprises collecting peripheral blood, obtaining asuspension of pure lymphocytes for cultivating test cultures with asuppression activator and without such activator, and further evaluatingproliferation levels (see Dutton R. W. Inhibitory and StimulatoryEffects of Concanavalin A on the Response of Mouse Spleen CellsSuspensions to Antigen. J. Exp. Med., v.138, p.1496-1505, 1973).

Nevertheless the known method requires a hardly available and expensiveforeign preparation, that is concanavalin A, as a suppression activator.

Known is a method for determining a suppressor component of human immunestatus which comprises collecting peripheral blood, obtaining asuspension of mononuclear cells (MNCs), dividing said suspension intotwo equal portions, cultivating MNCs from the first portion without asuppressor activator, and cultivating MNCs from the second portion witha suppressor activator, washing MNCs out of the culture medium andblocking proliferation, adding newly isolated MNCs from a normal donorinto each of abovementioned portions of MNCs, stimulating withphytohemagglutinin in equal proportions to obtain test cultures,cultivating them and further evaluating proliferation in said testcultures and determining suppression value on the basis of the raties ofproliferation levels in said test cultures (see By Lien Shov, Stanley A.Schwarts and Robert A. Good. Suppressor Cell Activity after ConcanavalinA treatment of Lymphocytes from Normal Donors. J. Exp. Med., 1976,v.143, n.5, p. 1100-1110).

Nevertheless said known method also requires a hardly available andexpensive foreign preparation, concanavalin A.

SUMMARY OF THE INVENTION

The main object of the present invention is to provide a less expensiveprocess for determining a suppressor activity of human immune status byusing a well available preparation which has an immune correctingactivity and does not cause allergic reactions.

Said object is achieved by use of trophoblastic β-I-glycoprotein (TBG)as a means for determining a suppressor component of human immunestatus, and the method of determination of a suppressor component ofhuman immune status which method comprises collecting a sample ofperipheral blood, obtaining a suspension of mononuclear cells (MNCs),dividing said suspension into two portions, cultivating the firstportion without a suppressor activator, and cultivating the second onewith a suppressor activator, washing MNCs out of the culture medium,blocking proliferation, adding into each of said MNC portions newlyisolated MNCs obtained from a normal donor, stimulating withphytohemagglutinin in equal proportions to produce test cultures,cultivating said cultures, further evaluating the proliferation of saidcultures and determining the suppression values based on the ratios ofthe levels of proliferation in test cultures. According to the presentinvention trophoblastic β-I-glycoprotein (TBG) is used in dosages from 3to 120 μg per 1 ml of MNC suspension.

The MNC suspension may be prepared from the cells obtained as a resultof separation in phycoll-urotrust single-step gradient, MNCs cultivationbeing carried out during 48 hours. Proliferation may be blocked bytreating MNCs with mitomycin C, and each of the test cultures may becultivated during 72 hours.

Experiments and clinical tests have shown new properties of TBG assuppressor activator useful for the determination of suppressorcomponent of human immune status.

PREFERRED EMBODIMENT OF THE INVENTION

At the first step MNC suspension is prepared from the cells obtained asa result of the separation in phycoll-urotrust single-stage densitygradient (Boyum's method).

Peripheral blood is taken from a patient by venipuncture and placedsimultaneously into the tubes containing heparine solution (1 ml ofblood=20-30 heparine units). Then the blood is diluted with Hankssolution in proportion 1:2 without Ca⁺⁺ and Mg⁺⁺ and layered ontophycoll-urotrust gradient (the density being 1.078).

Then centrifugation is carried out at 400 g during 30 min. MNCdispersion from interphase is placed into a centrifuge tube, Hankssolution without Ca⁺⁺ and Mg⁺⁺ is added, and 3 successivecentrifugations are carried out (10 in each) to wash the cells out ofphycoll-urotrust solution. After the third centrifugation MNC residue isresuspended in 1 ml of 199 medium, and the number of mononuclear cellsis counted using Goryajev camera.

At the second stage MNCs are divided into 2 equal portions, the firstportion is cultivated without a suppressor activator, and a second oneis cultivated with the suppressor activator, and trophoblasticβ-I-glycoprotein (TBG) is used as said activator.

MNCs are cultivated in penicillin vessels with 14.5 rubber plugs at 37°C. The culture medium is PPMI-1640 with 20% serum of IV (AB) group and300 mg of glutamine.

Each vessel contains 5×10⁶ cells in 2.0 ml of complete medium.

TBG in the dosages of 3-120 μg is added into the culture to inducesuppressor.

Cells are cultivated during 48 hours. Then MNCs are washed out of theculture medium and the proliferation is blocked by treating withmitomycin C (40 μg/ml during 30 min. at 37°). Then washing is performedthree times using 199 medium with 5% IV (AB) serum (chilled). Cellresidue is resuspended, the number of nucleus--containing cells iscounted, the percentage of cells viability is determined using 0.1%trypane blue solution, and the resulting suspension is diluted to therequired concentration. All operations are carried out separately forthe control cells, and for the TBG stimulated cells, and the siliconedishes are used to wash the cells.

At the next step newly isolated lymphocytes from a normal donor (whichlymphocytes are prestimulated with phytohemagglutinin (PHA) and used asresponding test cells) are added into each of the portions of controland TBG--stimulated lymphocytes in equal proportion (0.5×10⁶ :0.5×10⁶cells/ml) in order to obtain test cultures. The cultivation is carriedout during 72 hours. Then the proliferation of test cultures isevaluated using H³ -thymidine, and the suppression is evaluated by thedegree of proliferation decrease therein. Suppression index isdetermined using the following formula:

    SI=(1-pulse number/min. in test culture with TBG/pulse number/min. in test cultures without TBG)×100%

To evaluate the suppressor component in a normal donor a diagnosticstudy according to a known method (see By Lien Shov, Stanley A. Schwartsand Robert A. Good. Suppressor Cell Activity after Concanavalin ATreatment of Lymphocytes from Normal Donors. J.Exp.Med., 1976, v.143,n.5, p. 1100-1110), as well as a diagnostic study according to themethod of the present invention have been carried out using a group ofnormal donors (100 persons). Based on the results thus obtained thenormal value of T-suppressors activity under the induction withconcanavalin A is determined as 56.8% ±4%, while according to the methodof the present invention such value is 63.4% ±4.7%.

Further disclosure of the invention is provided in the followingexamples.

EXAMPLE 1

Patient V., 30, came to the hospital with the diagnosis of "disseminatedsclerosis, celebromedullary form" in acute stage. T-suppressors activityin peripheral blood under concanavalin A induction was 15%, which valuehad been determined according to the known method for evaluation of thesuppressor component of human immune status. SimultaneouslyT-suppressors activity in peripheral blood from the same patient wasdetermined using the method according to the present invention (underTBG induction), and the value thus obtained was 17%.

EXAMPLE 2

Patient S. (female), 40, came to the hospital with the diagnosis of"disseminated sclerosis, celebromedullary form" in acute stage.T-suppressors activity in peripheral blood under concanavalin Ainduction (determined according to the known method) was 16%.Simultaneously T-suppressors activity in peripheral blood in the samepatient was determined using the method according to the presentinvention (under TBG induction), and the value thus obtained was 19%.

150 patients suffering disseminated sclerosis and rheumatoid arthritishave been studied. It has been shown that T-suppressors activitydetermined by the method according to the invention corresponds to thevalues obtained by the known method.

Therefore high efficiency of TBG as a means for determining thesuppressor component of human immune status has been confirmed.

INDUSTRIAL APPLICABILITY

Abovementioned advantages of the method for determining a suppressorcomponent of human immune status according to the present invention, aswell as those of the means used therein, enable wide usage of saidmethod both for scientific purposes in clinical practice.

Also it should be noted that the use of concanavalin A for treatingabovementioned diseases is counterindicative due to toxicity thereof,while TBG (produced by human trophoblast) shows no toxicity and does notcause allergic reactions. This fact enables to use said preparation as adrug.

What is claimed is:
 1. A method for determining a suppressor componentof a patient's immune status comprising:(a) preparing first and secondsuspensions comprising mononuclear cells of the patient; (b) cultivatingthe mononuclear cells of the first suspension with a suppressoractivator consisting essentially of trophoblastic β-1 glycoprotein (TBG)in an amount effective to induce activation of suppressors in themononuclear cells of the first suspension; (c) cultivating themononuclear cells of the second suspension without a suppressoractivator; (d) blocking proliferation of the mononuclear cells in eachof the first and second suspensions; (e) adding responder cells to eachof the first and second suspensions in equal amounts so as to obtainrespective first and second test cultures; (f) cultivating the first andsecond test cultures under conditions that cause the responder cells toproliferate; and (g) determining the suppressor component of the immunesystem of the patient by comparing respective proliferation of theresponder cells in the first and second test cultures and assessing thesuppressor component based on the extent, if any, that proliferation ofthe responder cells of the first test culture is lower thanproliferation of the responder cells of the second test culture.
 2. Themethod as claimed in claim 1, wherein the responder cells aremononuclear cells from a normal donor that have been treated to causethem to proliferate.
 3. The method as claimed in claim 2, wherein theresponder cells have been treated with phytohemagglutinin.
 4. The methodas claimed in claim 2, wherein the first and second suspensions areprepared in step (a) by obtaining a sample of peripheral blood from thepatient and separating the mononuclear cells in a phycoll-urotrustsingle-step gradient.
 5. The method as claimed in claim 2, wherein step(d) comprises treating the mononuclear cells with mitomycin C.
 6. Themethod as claimed in claim 2, wherein in step (b) the mononuclear cellsof the first suspension are cultivated with 3 to 120 μg of TBG per 1 mlof the first suspension.
 7. The method as claimed in claim 6, whereinthe cultivating in step (b) is carried out for about 48 hours.
 8. Themethod as claimed in claim 2, wherein the cultivating in step (f) iscarried out for about 72 hours.
 9. In a method for determining asuppressor component of the immune status of a patient by comparing aproliferation of responder mononuclear cells in a first test culturewith a proliferation of responder mononuclear cells in a second testculture, said first and second test cultures comprising respective firstand second suspensions each of which comprises said responder cells andmononuclear cells of the patient, the mononuclear cells of the firstsuspension having been cultivated with a suppressor activator in themononuclear cells of the patient, the mononuclear cells of the secondsuspension having been cultivated without a suppressor activator, theimprovement wherein said suppressor activator is trophoblastic β-1glycoprotein (TBG).
 10. The method as claimed in claim 9, wherein theTBG is present in the first suspension in an amount of 3 to 120 μg per 1ml of the mononuclear cells of the patient.
 11. The method as claimed inclaim 10, wherein the responder cells are mononuclear cells from anormal donor that have been treated to cause them to proliferate. 12.The method as claimed in claim 10, wherein the responder cells have beentreated with phytohemagglutinin.
 13. The method as claimed in claim 11,wherein the first and second suspensions are prepared by obtaining asample of peripheral blood from the patient and separating themononuclear cells in a phycoll-urotrust single-step gradient.